The pathways of murine T-lymphocyte differentiation within the thymus, and after exit to the peripheral lymphoid tissues, will be investigated using both whole animal studies and cell culture models of the thymic environment. In addition the nature of the positive and negative selection operating to produce the particular H2-restriction specificity patterns of mature peripheral T cells will be investigated. Subsets of pre-thymocytes, thymocytes and peripheral T cells will be isolated on the basis of cell surface markers using specific antisera and flow cytofluorometry or cytotoxic procedures, as well as on the basis of physical properties using buoyant density, sedimentation velocity or electrophoretic methods. Precursor-product relationships amongst these subsets will be studied using in vivo labelling techniques (surface fluorescent labelling or DNA radiolabelling), as well as by isolating subsets and following their subsequent development in cell culture. Possible changes in the H2-restriction specificity of developing precursors of cytotoxic lymphocytes will be tested when the lymphoid and non-lymphoid elements of the cultures arise from mice differing at the major histocompatibility locus. The test system will involve a number of different haptens linked to appropriate target cells, and a new limiting dilution microculture system will be used. The results will be at the level of individual clones of cytotoxic T cells arising from single precursors, so individual precursors of a particular specificity can be enumerated.